3-(Heteroaryl acetamido)-2-oxo-azetidine-1-sulfonic acids derivatives as antibacterial agents

ABSTRACT

The present invention relates to novel Syn isomers of racemates and optical isomers of 3-(heteroaryl acetamido)-2-oxo-azetidine-1-sulfonic acids of the following formula: 
                 
 
and their use in treating infections caused by gram-negative pathogenic bacteria.

This application is a National Stage Application of InternationalApplication Number PCT/IB01/02115, published, pursuant to PCT Article21(2) which claims benefit of 60/232617, Sep. 14, 2000.

The present invention relates to novel Syn isomers of racemates andoptical isomers of 3-(heteroaryl acetamido)-2-oxo-azetidine-1-sulfonicacids and its use in treating the infections caused by gram-negativepathogenic bacteria.

BACKGROUND OF INVENTION

Bacteria are very adaptable microorganisms that possess the ability toadapt and to survive under adverse conditions. Doctors in hospitals andclinics around the world are losing the battle against an onslaught ofnew drug resistant bacterial infections including those caused byStaphylococci, Streptococci, Enterococci and Pseudomonas.

Bacterial resistance to the current antibiotics has been on a steep risedue to the alteration of the target, a change in the permeabilitypattern or by efflux of active ingredient and by deactivation of theantibiotic before reaching the active site.

The β-lactam antibiotics (penicillins, cephalosporins, monobactams andcarbapenems) are the most widely used group of antibiotics for thetreatment of many infectious diseases, because of proven clinicalefficacy and their excellent safety profile. Bacterial resistancetowards gram-positive pathogens against β-lactam antibiotics is mainlydue to the alteration of penicillin binding proteins (PBP's), efflux ofactive ingredient and deactivation of active ingredient. Whereasbacterial resistance towards gram-negative pathogens against β-lactamantibiotics in addition to those of the gram-positive pathogen, also aredue to changes in outer membrane permeability pattern.

To overcome the changes in outer membrane permeability, in recent yearsa number of β-lactam compounds (cephem and monobactam) containing aniron chelating catecholic and dihydroxypyridone groups have beenreported (29^(Th) ICAAC, Houston Tex., Sep. 18, 1989, abstract no. 355,356; 30^(th) ICAAC, Atlanta, Ga., Oct. 22, 1990, abstract no. 458;Antimicrobial Agents and chemotherapy 1991, 35, 104-110). The potentactivity of these compounds is due to their utilization of theTonB-dependent iron transport systems for transport across the bacterialouter membrane (Antimicrobial Agents and chemotherapy 1995, 39,613-619).

Monobactams are a class of antibacterial agents and have been used totreat infections caused by gram-negative microorganisms. CurrentlyAztreonam and Carumonam are in clinical use. Quinoxaline directlyattached to an oxime side chain of the monobactam nucleus is underdevelopment (Curr. Opin. Anti-infect. Drugs 1999, 1(1), 96-100;Antimicrobial Agents and Chemotherapy 1997, 41, 1010-1016). Furtherdihydroxypyridine through a methylene spacer attached to an oxime sidechain in the anti orientiation is reported as β-lactamase inhibitor(U.S. Pat. No. 5,888,998 (1999)).

The present invention describes a class of compound in which adihydroxypyridone group is directly or through a suitable spacerattached to an oxime side chain in a monobactam nucleus and its use totreat gram-negative infections, particularly those caused byPseudomonas. Pseudomonas aeruginosa continues to be a very frequentopportunistic pathogen, capable of causing a wide variety of infectionsin the immunocompromised patient. These infections are often associatedwith significant morbidity and are difficult to treat.

SUMMARY OF THE INVENTION

It is an objective of the present invention to provide novel Syn isomersof racemates and optical isomers of 3-(heteroarylacetamido)-2-oxo-azetidine-1-sulfonic acids of formula I havingantibacterial activity against gram-negative pathogenic bacteria,particularly Pseudomonas strains.

It is a further object of the invention to provide pharmaceuticalcompositions comprising the compound of formula I with apharmaceutically acceptable carrier or diluent.

It is an additional object of the invention to provide a method fortreatment of bacterial infections caused by gram-negative pathogenicbacteria including Pseudomonas.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided novel Synisomers of recemates and optical isomers of 3-(heteroarylacetamido)-2-oxo-azetidine-1-sulfonic acids of formula I

Wherein

-   M is a hydrogen or a pharmaceutically acceptable salt forming    cation;-   X is CH, N or C-halo;-   R is C₁-C₃ allyl which is unsubstituted or substituted with at least    one of (a) a halogen atom (b) OR₅ wherein R₅ is hydrogen, CONH₂ or    2,5-dihydroxy-4-oxo-1,4-dihydro-pyridin-2-yl-carbonyl and wherein a    C₁ alkyl may not be substituted with both a halogen atom and OR₅.-   R₁ and R₂ independently are OH, COOH, CONH₂, optionally substituted    phenyl or C₁-C₃ alkyl; or-   R₁ and R₂ together are —O—CH═CH—CH₂—, —O—CH₂—CH₂—O—, —CH₂—CH₂—CH₂—,    —CH₂—CH₂—CH₂—CH₂—, —CH═CH—CH═CH— or —CH═C(OH)—C(OH)═CH— which    together with the carbon atoms to which they are bound form a 5    membered or six membered cyclic ring-   R₃ and R₄ independently are hydrogen, optionally substituted C₁-C₃    alkyl, optionally substituted phenyl or C₃-C₆ cycloalkyl;-   R₃ and R₄ together are C₃-C₆ cycloalkyl.

As used herein, the term “C₁-C₃ alkyl” means a straight or branch chainalkyl having 1-3 carbon atom selected from methyl, ethyl, propyl andisopropyl.

As used herein, the term “C₃-C₆ cycloalkyl” means a saturated alicyclicmoiety having 3-6 carbon atoms selected from cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl.

As used herein, the term “halogen atom” means fluorine, chlorine, orbromine.

As used herein, the term “substituted” as applied to a group meanssubstituted with 1, 2 or 3 substituents selected from OH, NH₂,dimethylamino, a halogen atom, OCH₃, COOH, CONH₂, NO₂ or CN.

As used herein, the term “racemate” means the mixture ofdiastereoisomers having zero optical rotation of the molecule of formulaI.

As used herein, the term “optical isomers” means pure single R and Sdiastereoisomers at the asymmetric carbon atoms present in the moleculeof formula I.

As used herein the term “pharmaceutically acceptable salt formingcation” means alkali metals (e.g. Sodium, Potassium), alkaline earthmetals (e.g. Calcium, Magnesium), organic bases (e.g. triethylamine,ethanolamine, n-methylmorpholine) or basic amino acids (e.g. lysine,arginine, orithine or histidine). Moreover when M is hydrogen in formulaI, it can form zwitterions (inner salt or internal salt) by interactingwith a basic nitrogen atom present in the molecule of formula I.

In accordance with the preferred embodiment of the present invention,there is provided novel Syn isomers of racemates and optical isomers of3-(heteroaryl acetamido)-2-oxo-azetidine-1-sulfonic acids and of formulaI

Wherein

-   M is a hydrogen or pharmaceutically acceptable salt forming cation;-   X is CH;-   R is CH₃, CH₂F or CH₂OCONH₂.-   R₁ is OH-   R₂ is Hydrogen;-   R₁ and R₂ together is —CH═C(OH)—C(OH)═CH— which forms six member    cyclic ring-   R₃ and R₄ independently is hydrogen;-   R₃ and R₄ together is cyclopropyl;

As used herein, the term “racemate” means the mixture ofdiastereoisomers having zero optical rotation of the molecule of formulaI.

As used herein, the term “optical isomers” means pure single R and Sdiastereoisomers at the asymetric carbon atoms present in the moleculeof formula I.

As used herein the term “pharmaceutically acceptable salt formingcation” means alkali metals (e.g. Sodium, Potassium). Moreover when M ishydrogen in formula I, it can form zwitterion (inner salt or internalsalt) by interacting with a basic nitrogen atom present in the moleculeof formula I.

The compounds of this invention can be used to treat bacterialinfections caused by gram-negative bacteria, including but not limitedto Pseudomonas E. eloaecae, C. freundii, M. Morganii, K. paeumoniae, andE. Coli alone or in combination with other drugs in mammals includinghumans. The compounds may be administered in pharmaceutical dosage formsincluding parenteral preparation such as injections, suppositories,aerosols and the like, and oral preparations such as tablets, coatedtablets, powders, granules, capsules, liquids and the like. The abovepreparations are formulated in manners well known to the art.

For the formulation of solid preparations for oral administration, anexcipient, and if desired, a binder, disintegrator, lubricant, coloringagent, corrigent, flavor etc. are added to the compound of theinvention, and then tablets, coated tablets, granules, powders, capsulesor the like are prepared in a conventional manner.

For the formulation of injections, a pH adjusting agent, buffer,stabilizer, isotonic agent, local anesthetic or the like is added to theactive ingredient of the invention, and injections for subcutaneous,intramuscular or intravenous administration can be prepared in theconventional manner.

For the formulation of suppositories, a base, and if desired,surfactants are added to the active ingredient of the invention, and thesuppositories are prepared in a conventional manner.

The excipients useful for solid preparations for oral administration arethose generally used in the art, and the useful examples are excipientssuch as lactose, sucrose, sodium chloride, starches, calcium carbonate,kaolin, crystalline cellulose, methyl cellulose, glycerin, sodiumalginate, gum arabic and the like, binders such as polyvinyl alcohol,polyvinyl ether, polyvinyl pyrrolidone, ethyl cellulose, gum arabic,schellac, sucrose, water, ethanol propanol, carboxymethyl cellulose,potassium phosphate and the like, lubricants such as magnesium stearate,talc and the like, and further include additives such as usual knowncoloring agents, disintegrators and the like. Examples of bases usefulfor the formulation of suppositories are oleaginous bases such as cacaobutter, polyethylene glycol, lanolin, fatty acid triglycerides, witepsol(trademark, Dynamite Nobel Co. Ltd.) and the like. Liquid preparationsmay be in the form of aqueous or oleaginous suspension, solution, syrup,elixir and the like, which can be prepared by a conventional way usingadditives.

The amount of the compound I of the invention incorporated into thepharmaceutical composition of the invention varies with the dosage form,solubility and chemical properties of the compound, administrationroute, administration scheme and the like. Preferably the amount isabout 1 to 25 w/w % in the case of oral preparations, and about 0.1 to 5w/w % in the case of injections which are parenteral preparations.

The dosage of the compound I of the invention is suitably determineddepending on the individual cases taking symptoms, age and sex of thesubject and the like into consideration. Usually the dosage in the caseof oral administration is about 50 to 1500 mg per day for an adult in 2to 4 divided doses, and the dosage in the case of injection, forexample, by intravenous administration is 2 ml (about 1 to 100 mg) whichis administered once a day for adults wherein the injection may bediluted with physiological saline or glucose injection liquid if sodesired, and slowly administered over at least 5 minutes. The dosage incase of suppositories is about 1 to 1000 mg which is administered onceor twice a day at an interval of 6 to 12 hours wherein the suppositoriesare administered by insertion into the rectum.

The compounds of the present invention having the formula I can beprepared by reacting 3-amino-azetidine-2-one sulfonic acid of formula(II) with heteroaryl carboxylic acid of formula III followed bydeprotection of the protecting group.

Certain derivatives of general formula IV were prepared by coupling of3-amino-azetidine-2-one sulfonic acid (II) with a heteroaryl carboxylicacid (III) in presence of dicyclohexylcarbodiimide (DCC) or with an acidchloride of compound (III) in presence of base, or with an activatedester of compound (III) within the skill of the arts.

Alternatively, compounds of formula I can also be prepared as follows:

The preparation of compound II (R=CH₃) was carried out by following thesynthetic scheme 2 as described in J. Org. Chem. 1982, 47, 5160-5167.

The preparation of compound II (R=CH₂F, CH₂OCONH₂) was carried out byfollowing the synthetic scheme 3 from common intermediate compound V asdescribed in J. Antibiotics 1983, 36, 1201-1204 and J. Antibiotics 1985,38, 346-357.

The common intermediate compound V was prepared by following thesynthetic route as described in scheme 4. The distereoisimers ofcompound VI are separated by optical resolution methods (J. Antibiotics1985, 38, 346).

The preparation of compounds III was done by reacting 2-heteroaryl-2-oxoacetic acid (VII) with O-heteroaryl hydroxyl amine (VIII) at roomtemperature and afforded exclusively the syn-isomer. The preparation ofcompound VIII was done as described in Scheme 5 starting fromheteroarylmethanol (J. Antibiotics 1990, 43, 189-198).

In the above descriptions (scheme 1-5), the reactants are reactedtogether with a suitable solvent at elevated or low temperatures forsufficient time to allow the reaction to proceed to completion. Thereaction conditions will depend upon the nature and reactivity of thereactants. Wherever a base is used in a reaction, they are selected fromtriethylamine, tributylamine, trioctylamine, pyridine,4-dimethylaminopyridine, diisopropylethylamine,1,5-diazabicyclo[4,3,0]non-5-ene, 1,8-diazabicyclo[5,4,0]undec-7-ene,sodium carbonate, sodium bicarbonate, potassium carbonate, potassiumbicarbonate or cesium carbonate.

The deprotection of the protective group is carried out either byhydrogenation or by hydrolysis with appropriate acids such ashydrochloric acid, trifluoroacetic acid or acetic acid in solvent suchas methanol, ethanol, propanol or ethyl acetate. The hydrogenationreaction is usually carried out in the presence of a metal catalyst,such as Pd, Pt, or Rh, under normal pressure to high pressure.

The solvents of choice for the reaction are selected based upon thereactants used and from such solvents as benzene, toluene, acetonitrile,tetrahydrofuran, ethanol, methanol, chloroform, ethyl acetate, methylenechloride, dimethyl formamide, dimethyl sulfoxide, hexamethyl phosphorictriamide, or the like. Solvent mixtures may also be utilized.

Reaction temperatures would generally range from between −70° C. to 150°C. The preferred molar ratio of reactants is 1:1 to 1:5. The reactiontime range from 0.5 to 72 hours, depending on the reactants.

The present invention is further illustrated by the followingnon-limiting examples.

EXAMPLE 1(3S)-trans-3-[(2-Amino)thiazol-4-yl)-(Z)-2-{(1,5-dihydroxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-methyl-2-oxoazetidine-1-sulfonic acid, sodiumsalt

Step 1: 1,5-Dibenzhydryloxy-2-(N-phthalimido)oxymethyl-4-pyridone

A solution of 1,5-dibenzhydryloxy-2-hydroxymethyl-4-pyridone (20.0 g,0.041 mol) and N-hydroxyphthalimide (6.64 g, 0.048 mol) in a mixture ofTHF (200 ml) and dry DMF (200 ml) was treated with triphenyl phospheneunder nitrogen and cooled to 0° C. The reaction mixture was then addedwith diethyl azodicarboxylate dropwise over 10 min., stirred at 0° C.for 1 h then diluted with ethyl acetate and water. The organic layer wasseparated, washed with water and brine, dried over sodium sulfate,filtered and evaporated in vacuo. The crude product obtained waspurified by silica gel column chromatography using a gradient mixture ofEA: Hexane (1:2 to 1:0) to give the pure title compound.

Yield: 19.0 g, 73% ¹HNMR (DMSO-d₆): δ 4.78(s, 2H), 6.24(s, 1H), 6.29(s,1H), 6.46(s, 1H), 7.18-7.38(m, 20H), 7.62(s, 1H), 7.85(s, 4H).Step 2:2-(2-Tritylamino)-thiazol-4-yl)-(Z)-2-[1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy]-imino acetic acid.

A solution of 1,5-dibenzhydryloxy-2-(N-phthalimido)oxymethyl-4-pyridone(10 g, 15.8 mmol) in ethanol (98%, 100 ml) was treated with hydrazine(0.76 ml). The mixture was heated to reflux for 1 h. and cooled to RT.The suspension thus obtained was filtered and the filtrate wasevaporated to dryness and was treated with chloroform. The solid thusseparated was filtered off, the mother liquors were concentrated and theresidue obtained was dissolved in ethanol (98%) then treated with asolution of 2-oxo-2-[(N-tritylamino)thiazol-5-yl]acetic acid (6.38 g) inchloroform. The reaction mixture was stirred at room temperature for 18h and evaporated in vacuo. The residue obtained was dissolved in ethylacetate and diluted with hexanes. The solid separated was filtered anddried to give2-(2-tritylamino)thiazol-4-yl)-(Z)-2-[1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy]imino acetic acid.

Yield: 11.2 g, 79% ¹HNMR (DMSO-d₆): δ 4.62(s, 2H), 6.03(s, 1H), 6.28(s,1H), 6.40(s, 1H), 6.66(s, 1H), 7.18-7.35(m, 35H), 7.48(s, 1H), 8.64(s,1H).Step 3:(3S)-trans-3-[2-(2-Tritylamino)thiazol-4-yl)-(Z)-2-{(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-methyl-2-oxoazetidine-1-sulfonic acid.

A mixture of (3S)-trans-3-amino-4-methyl-2-oxoazetidine-1-sulfonic acid[7.30 g, 40.52 mmol, J. Org. Chem., 47, 5160, (1982)],2-(2-tritylamino)thiazol-4-yl)-(Z)-2-[1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy]imino acetic acid (from step 36.50 g, 40.51 mmol), DCC (9.15 g,44.34 mmol) and 1-hydroxybenzotriazole (5.47 g, 40.5 mmol) in dry DMF(400 ml) was stirred at room temperature for 30 min. and to this mixtureNaHCO3 (3.40 g, 40.52 mmol) was added. The mixture was stirred undernitrogen at room temperature over night and filtered. The mother liquorwas evaporated in vacuo to remove DMF and the residue obtained wasdissolved in ethyl acetate and distilled water and adjusted to pH ˜3.The organic layer was dried over anhydrous sodium sulfate, filtered andevaporated in vacuo.

The product thus obtained was purified over HP-20 column chromatographyusing a gradient mixture of water:acetonitrile (1:0 to 1:9) to give thetitle compound.

Silica gel column chromatography using a gradient mixture of ethylacetate: methanol (1:0 to 9:1) gave the title compound

Yield: 37.00 g, 85.9% ¹HNMR (DMSO-d₆): δ 1.29(d, 3H, J=6.0 Hz),3.54-3.61(m, 1H), 4.30-4.35(m, 1H), 4.70(s, 2H), 5.98(s, 1H), 6.29(s,2H), 6.71(s, 1H), 7.25-7.35(m, 35H), 7.51(s, 1H), 8.83(s, 1H), 9.39(d,1H, J=7.7 Hz).

Step 4:(3S)-trans-3-[(2-Amino)thiazol-4-yl)-(Z)-2-{(1,5-dihydroxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-methyl-2-oxoazetidine-1-sulfonic acid.

A suspension of(3S)-trans-3-[-2-(2-tritylamino)thiazol-4-yl)-(Z)2-{(1,5-dibenzhydryloxy-4-pyridon-2-yl

methoxy)imino}acetamido]-4-methyl-2-oxoazetidine-1-sulfonic acid (5.00g, 4.703 mmol) in dry anisole (14 ml) at −10° C., under nitrogen wastreated with trifluoroacetic acid (25 ml) and stirred at 0° C. for 2hrs. The solvents were evaporated under reduced pressure and the residuewas triturated with a mixture of ether-hexane and ethyl acetate (1:1:1).The solid thus obtained was filtered, washed with a mixture ofether-hexane and ethyl acetate (1:1:1) to give a solid. The above solidwas further purified by HP-20 column chromatography using a gradientmixture of distilled water and acetonitrile (1:0 to 9:1) and theappropriate fractions were lyophilized to give the title compound.

Yield: 2.7 g, 92%; mp: 200° C. decomp. ¹HNMR (DMSO d₆): δ 1.41(d, 3H,J=6.2 Hz), 3.70-3.80(m, 1H), 4.46(dd, 1H, J=2.4 Hz and 5.2 Hz), 5.30(s,2H), 6.85(s, 1H), 7.05(s, 1H), 7.35(br, s, 2H), 8.17(s, 1H), 9.50(d, 1H,J=7.7 Hz).Step 5:(3S)-trans-3-[(2-Amino)thiazol-4-yl)-(Z)-2-{(1,5-dihydroxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-methyl-2-oxoazetidine-1-sulfonic acid, sodiumsalt.

A suspension of(3S)-trans-3-[(Z)-(2-amino)thiazol-4-yl)-2-{(1,5-dihydroxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-methyl-2-oxoazetidine-1-sulfonic acid (1.30g, 2.66 mmol) in distilled water (15 ml) was cooled to ˜5-6° C. andNaHCO3 (s, 0.223 g, 2.654 mmol) was added in portions with stirring. Theclear solution thus obtained within 10 min. was filtered and lyophilizedto give(3S)-trans-3-[{(2-amino)thiazol-4-yl)-(Z)-2-{(1,5-dihydroxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-methyl-2-oxoazetidine-1-sulfonic acid, sodiumsalt.

Yield: 1.32 g, 97.13%. ¹HNMR (DMSO-d₆): δ 1.42(d, 3H, J=6.1 Hz),3.70-3.80(m, 1H), 4.48-4.53(m, 1H), 5.13(s, 2H), 6.64(s, 1H), 6.79(s,1H), 7.24(br, s, 2H), 7.68(s, 1H), 9.52(d, 1H, J=7.0 Hz).

EXAMPLE 23-[2-(2-Aminothiazol-4-yl)-(Z)-2-{(1,5-dihydroxy-4-pyridon-2-ylmethoxy)imino}-acetamido]-4-carbamoyloxymethyl-2-azetidinone-1-sulfonicacid, sodium salt

Step 1:3-[2-(2-Tritylamino)-thiazol-4-yl)-(Z)-2-{(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-carbamoyloxymethyl-2-azetidinone

A solution of2-(2-tritylamino)-thiazol-4-yl)-(Z)-2-[1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy]imino acetic acid (0.34 g, 0.377 mmol) in dry DMF (20 ml) wastreated with DCC (0.078 g, 0.377 mmol) and 1-hydroxybenzotriazole (0.050g, 0.0377 mmol). The mixture was stirred under nitrogen at roomtemperature for 30 min. and to this mixture NaHCO₃ (0.032 g, 0.377mmole) and 3-amino-4-carbamoyloxymethyl-2-azetidinone (0.06 g, 0.377mmol) in DMF (5 ml) was added. The reaction mixture was stirred at roomtemperature for 18 hrs, and DMF was removed in vacuo. The product thusobtained was purified by silica gel column chromatography by a gradientmixture of ethyl acetate and methanol (10:1 to 9.5:0.5) to give thetitle compound.

Yield: 0.2 g, 97.13% ¹HNMR (DMSO-d₆): δ 3.80-3.92(m, 2H), 3.97-4.05(m,1H), 4.70(s, 2H), 5.17-5.25(m, 1H), 6.00(s, 1H), 6.31(s, 1H), 6.53(br,s, 2H), 6.74(s, 1H), 7.24-7.38(m, 35H), 7.58(s, 1H), 8.50(s, 1H),8.80(s, 1H), 9.29(d, 1H, J=9.0 Hz).Step 2:3-[{2-(2-Tritylamino)thiazol-4-yl)}-(Z)-2-{(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-carbamoyloxymethyl-2-azetidinone-1-sulfonicacid

A solution of3-[{2-(2-tritylamino)thiazol-4-yl)}-(Z)-2-{(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-carbamoyloxymethyl-2-azetidinone (0.25 g,0.244 mmol) in pyridine (2 ml) was treated with sulfur trioxide-pyridinecomplex (0.153 g, 0.96 mmol) and the mixture was heated at 70° C. for 45min. The reaction mixture was cooled to RT, treated with diethyl etherand the solid was filtered, washed with distilled water followed byether and dried to giveciss-3-[{2-(2-tritylamino)thiazol-4-yl)}-(Z)-2-{(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-carbamoyloxy methyl-2-azetidinone-1-sulfonicacid.

Yield: 0.23 g, 85%Step 3:3-[2-(2-Tritylamino)-thiazol-4-yl)-(Z)-2-{(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-carbamoyloxymethyl-2-azetidinone-1-sulfonicacid, sodium salt.

A suspension of3-[2-(2-tritylamino)-thiazol-4-yl)-(Z)-2-{(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-carbamoyloxymethyl-2-azetidinone-1-sulfonicacid (0.390 g, 0.353 mmol) in distilled water (10 ml) was treated withNaHCO₃ (s, 0.050 g, 0.595 mmol) and stirred at RT for 30 min. and theclear solution was lyophilized. The solid obtained was purified by HP-20column chromatography using a gradient mixture of dd. Water andacetonitrile (1:0 to 3:7), and the appropriate fractions werelyophilized to give the to give3-[2-(2-tritylamino)-thiazol-4-yl)-(Z)-2-{(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-carbamoyloxymethyl-2-azetidinone-1-sulfonicacid, sodium salt.

Yield: 0.21 g, 52%Step 4: 3-[2-(2-Aminothiazol-4-yl)-(Z)-2-{(1,5-dihydroxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-carbamoyloxymethyl-2-azetidinone-1-sulfonicacid.

A suspension of3-[2-(2-tritylamino)thiazol-4-yl)-(Z)-2-{(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-carbamoyloxymethyl-2-azetidinone-1-sulfonicacid, sodium salt (0.8 g, 0.874 mmol) in anisole (5 ml) under nitrogenatmosphere was cooled to ˜0° C. and treated with trifluoroacetic acid(25 ml) and the mixture was stirred at less than 10° C. for 2 hrs andtreated with ether. The solid separated was filtered, washed withacetone and dissolved in a mixture of acetonitrile/dd: H₂O and freezedried to give the title compound.

Yield: 0.34 g, 89%; mp: 190° decomp. ¹HNMR (DMSO-d₆): δ 3.90-4.30(m,3H), 5.22-5.40(m, 5H), 6.50(br, s, 2H), 6.82(s, 1H), 6.95(s, 1H),7.33(br, s, 2H), 8.00(s, 1H), 9.45(d, 1H, J=7.5 Hz).Step 5: 3-[2-(2-Aminothiazol-4-yl)-(Z)-2-{(1,5-dihydroxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-carbamoyloxymethyl-2-azetidinone-1-sulfonicacid, sodium salt

NaHCO₃ (s, 6 mg, 0.073 mmol) was added to a suspension of3-[2-(2-Aminothiazol-4-yl)-(Z)-2-{(1,5-dihydroxy-4-pyridon-2-yl-methoxy)imino}-acetamido]-4-carbamoyloxymethyl-2-azetidinone-1-sulfonicacid (40 mg, 0.073 mmol) in distilled water. After stirring for 5 min.the mixture was freeze dried to give the title compound as a solid.

Yield: 30 mg, 71% ¹HNMR (DMSO-d₆): δ 4.03-4.15(m, 2H), 4.20-4.33(m, 1H),5.12(s, 2H), 5.26-5.37(m, 1H), 6.54(br, s, 2H), 6.70(s, 1H), 6.77(s,1H), 7.24(br, s, 2H), 7.72(s, 1H), 9.38(d, 1H, J=7.5 Hz).

EXAMPLE 33-[2-(2-Aminothiazol-4-yl)-(Z)-2-{(1,5-dihydroxy-4-pyridon-2-yl-methoxy)imino}-acetamido]-4-fluoromethyl-2-azetidinone-1-sulfonicacid, sodium salt

Step 1:3-[2-(2-Tritylamino)-thiazol-4-yl)-(Z)-2-{(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-fluoromethyl-2-azetidinone-1-sulfonic acid.

A solution of3-(N-benzyloxycarbonyl)amino-4-fluoromethyl-2-azetidinone-1-sulfonicacid, tetrabutyl ammonium salt (0.5 g, 0.89 mmol) in DMF (20 ml) wastreated with Pd—C (0.3 g) and the suspension was hydrogenated at 50 psiover 5 hrs. The suspension was filtered through celite and to thefiltrate was added DCC (0.18 g, 0.89 mmol), 1-HBT (0.12 g, 0.89 mmol)followed by2-{(2-tritylamino)-thiazol-4-yl}-(Z)-2-[1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy]imino acetic acid (0.4 g, 0.89 mmol). The reaction mixture wasstirred at RT for 18 hrs and evaporated in vacuo. The residue wasdissolved in acetone treated with potassium nonafluoroborate (0.6 g) inacetone and stirred for a further 18 hrs. The solvents were evaporatedand the residue was treated with a mixture of Ethyl acetate-Ether-Hexane(1:1:1). The solid separated was filtered and purified by silica gelcolumn chromatography using a gradient mixture of Ethyl acetate andmethanol (10:1 to 9:1) to give the title compound.

Yield: 0.22 g, 42.8% ¹HNMR DMSO-d₆): δ 4.00-4.20(m, 2H), 4.40-4.50(m,1H), 4.67(s, 2H), 5.16-5.24(m, 1H), 6.00(s, 1H), 6.32(s, 1H), 6.37(s,1H), 6.67(s, 1H), 7.27-7.43(m, 35H), 7.63(s, 1H), 8.85(s, 1H), 9.46(d,1H, J=9.0 Hz).Step 2: 3-[2-(2-Aminothiazol-4-yl)-(Z)-2-{(1,5-dihydroxy-4-pyridon-2-ylmethoxy) imino}-acetamido]-4-fluoromethyl-2-azetidinone-1-sulfonic acid,sodium salt

A suspension of3-[2-(2-tritylamino)thiazol-4-yl)-(Z)-2-{(1,5-dibenzhydryloxy-4-pyridon-2-ylmethoxy)imino}acetamido]-4-fluoromethyl-2-azetidinone-1-sulfonic acid(0.5 g, 0.46 mmol) in anisole (2 ml), under nitrogen at −10° C. wastreated with trifluoroacetic acid (20 ml) and stirred at 5-10° C. for 2hrs. The reaction mixture was evaporated in vacuo and the residue wastriturated with a mixture of ether:ethyl acetate and hexanes (1:1:1).The solid separated was filtered, dissolved in acetonitrile, watermixture and freeze dried. The crude product obtained was furtherpurified by HP-20 column chromatography using a gradient mixture ofdd.H₂O and acetonitrile (1:0 to 9.4:0.6) to give3-[2-(2-aminothiazol-4-yl)-(Z)-2-{(1,5-dihydroxy-4-pyridon-2-ylmethoxy)imino}-acetamido]-4-fluoromethyl-2-azetidinone-1-sulfonic acid.

Yield: 80 mg, 34%; M.pt.: 200° C. decomp. ¹HNMR (DMSO-d₆): δ3.83-4.35(m, 2H), 4.47-4.63(m, 1H), 4.68-4.85(m, 1H), 5.28(s, 2H),6.29(s, 1H), 7.03(s, 1H), 7.30(br, s, 3H), 8.12(s, 1H), 9.45(d, 1H,J=8.1 Hz).Step 3: 3-[2-(2-Aminothiazol-4-yl)-(Z)-2-{(1,5-dihydroxy-4-pyridon-2-ylmethoxy) imino}-acetamido]-4-fluoromethyl-2-azetidinone-1-sulfonic acid,sodium salt

NaHCO₃ (s, 13 mg, 0.155 mmol) was added to a suspension of3-[2-(2-Aminothiazol-4yl)-(Z)-2-{(1,5-dihydroxy-4-pyridon-2-yl-methoxy)imino}-acetamido]-4-fluoromethyl-2-azetidinone-1-sulfonicacid (80 mg, 0.158 mmol) in distilled water. The mixture was stirred for5 min. and freeze dried to give3-[2-(2-aminothiazol-4-yl)-(Z)-2-{(1,5-dihydroxy-4-pyridon-2-ylmethoxy)imino}-3-acetamido]-4-fluoromethyl-2-azetidinone-1-sulfonicacid, sodium salt

Yield: 75 mg, 89% ¹HNMR (DMSO-d₆): δ 3.83-4.30(m, 2H), 4.47-4.64(m, 1H),4.73-4.84(m, 1H), 5.13(s, 2H), 5.30(s, 1H), 6.55(s, 1H), 6.74(s, 1H),7.27(br, s, 2H), 7.57(s, 1H), 9.57(br, s, 1H).

Test for Antibacterial Activity

The compounds of the present invention were tested for minimuminhibitory concentration (MIC) against the bacteria listed in Table-1according to the standard microbroth dilution method as described inNCCLS document. The minimum inhibitory concentration is expressed inμg/ml.

TABLE 1 Antibacterial activity of the compounds of formula I Testedcompounds (MIC in μg/ml) Organisms 1 2 3 Aztreonam Carumonam E. coli0.06 0.06 0.06 0.06 0.06 TEM-2 K pneumoniae 0.13 0.06 0.06 0.06 0.06K-1150 M Morganii 0.06 0.13 0.06 0.06 0.06 K 1250 C. freundii 0.13 0.500.50 0.13 0.06 K 500 E. cloaacae 0.06 0.25 2.0 0.13 0.06 S 480-2 P.aeruginosa 0.06 0.06 0.06 32 8.0 CL 1267 P. aeruginosa 0.13 0.13 0.13 328 S 1598 P. aeruginosa 4.0 128 32 64 32 PD 2721

1. A compound selected from the group consisting of a Syn isomer of aracemate and an optical isomer of 3-(heteroarylacetamido)-2-oxo-axetidine-1-sulfonic acid and of formula I

Wherein M is a hydrogen or pharmaceutically acceptable salt formingcation; X is CH, N or C-halogen; R is C₁-C₃ alkyl which is unsubstitutedor substituted with at least one of (a) a halogen atom (b) OR₅ whereinR₅ is hydrogen, CONH₂ provided that, when R is methyl, it cannot besubstituted with both a halogen atom and OR₅; R₁ and R₂ independentlyare OH, COOH, CONH₂, NH₂, dimethylamino, OCH₃, NO₂, CN, optionallysubstituted phenyl or C₁-C₃ alkyl; or R₁ and R₂ together are—O—CH═CH—CH₂—, —O—CH₂—CH₂—O—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—,—CH═CH—CH═CH— or —CH═C(OH)—C(OH)═CH— which together with the carbonatoms to which they are bound form a 5 membered or six membered cyclicring; and R₃ and R₄ independently are hydrogen, optionally substitutedC₁-C₃ alkyl, optionally substituted phenyl or C₃-C₆ cycloalkyl; or R₃and R₄ together with the C to which they are attached are C₃-C₆cycloalkyl.
 2. The compound of formula I as set forth in claim 1,wherein at least one of R, R₁, R₂, R₃, and R₄ are selected from thegroup consisting of methyl, ethyl, propyl and isopropyl.
 3. The compoundof formula I as set forth in claim 1, wherein R3 and R4 areindependently a saturated alicyclic moiety having 3-6 carbon atomsselected from the group consisting of cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl.
 4. The compound of formula I as set forth inclaim 1, wherein the halogen atom is selected from the group consistingof fluorine, chlorine, and bromine.
 5. The compound of formula I as setforth in claim 1, wherein the substitutents are 1, 2 or 3 substituentsselected from OH, NH2, dimethylamino, halogen atom, OCH₃, COOH, CONH₂,NO₂ or CN.
 6. A compound of formula I as set forth in claim 1, whereinthe pharmaceutically acceptable salt forming cation is selected from thegroup consisting of alkali metals, alkaline earth metals, organic basesor basic amino acids.
 7. A compound of formula I

Wherein M is a hydrogen or pharmaceutically acceptable salt formingcation; X is CH; R is CH₃, CH₂F or CH₂OCONH₂; R₁ is OH; R₂ is Hydrogen;or R₁ and R₂ together are —CH═C(OH)—C(OH)═CH— which, together with thecatons to which R₁ and R₂ are bound, forms a six member cyclic ring; andR₃ and R₄ independently are hydrogen; or R₃ and R₄ together with the Cto which they are attached are cyclopropyl.
 8. A pharmaceuticalcomposition suitable for the treatment of bacterial infections inmammals comprising a compound selected from the group consisting of aSyn isomer of a racemate and an optical isomer of 3-(heteroarylacetamido)-2-oxo-axetidine-1-sulfonic acid and of formula I

Wherein M is a hydrogen or pharmaceutically acceptable salt formingcation; X is CH, N or C-halogen; R is C₁-C₃ alkyl which is unsubstitutedor substituted with at least one of (a) a halogen atom (b) OR₅ whereinR₅ is hydrogen, CONH₂ or2,5-dihydroxy-4-oxo-1,4-dihydro-pyridin-2-yl-carbonyl, wherein C₁ cannotbe substituted with both a halogen atom and OR₅; R₁ and R₂ independentlyare OH, COOH, CONH₂, NH₂, dimethylamino, OCH₃, NO₂, CN, optionallysubstituted phenyl or C₁-C₃ alkyl; or R₁ and R₂ together are—O—CH═CH—CH₂—, —O—CH₂—CH₂—O—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—,—CH═CH—CH═CH— or —CH═C(OH)—C(OH)═CH— which together with the carbonatoms to which they are bound form a 5 membered or six membered cyclicring; and R₃ and R₄ independently are hydrogen, optionally substitutedC₁-C₃ alkyl, optionally substituted phenyl or C₃-C₆ cycloalkyl; or R₃and R₄ together are C₃-C₆ cycloalkyl; or a pharmaceutically acceptablesalt of said compound; and wherein said composition further comprises apharmaceutically acceptable carrier.
 9. A method of treating a bacterialinfection in a subject in need of such treatment comprisingadministering to said subject in need of such treatment atherapeutically effective amount of a compound selected from the groupconsisting of a Syn isomer of a racemate and an optical isomer of3-(heteroaryl acetamido)-2-oxo-axetidine-1-sulfonic acid and of formulaI

Wherein M is a hydrogen or pharmaceutically acceptable salt formingcation; X is CH, N or C-halogen; R is C₁—C₃ alkyl which is unsubstitutedor substituted with at least one of (a) a halogen atom (b) OR₅ whereinR₅ is hydrogen, or, CONH₂, provided that, when R is methyl, it cannot besubstituted with both a halogen atom and OR₅; R₁ and R₂ independentlyare OH, COOH, CONH₂, NH₂, dimethylamino, OCH₃, NO₂, CN, optionallysubstituted phenyl or C₁-C₃ alkyl; or R₁ and R₂ together are—O—CH═CH—CH₂—, —O—CH₂—CH₂O—, —CH₂—CH₂—CH₂—, —CH₂CH₂—CH₂CH₂—,—CH═CH—CH═CH— or —CH═C(OH)—C(OH)═CH— which together with the carbonatoms to which they are bound form a 5 membered or six membered cyclicring; and R₃ and R₄ independently are hydrogen, optionally substitutedC₁-C₃ alkyl, optionally substituted phenyl or C₃-C₆ cycloalkyl; or R₃and R₄ together with the C to which they are attached are C₃-C₆cycloalkyl.
 10. The composition, according to claim 8, wherein saidcomposition is formulated for oral use and comprises about 1 to 25 w/w %of a compound selected from the group consisting of a Syn isomer of aracemate and an optical isomer of 3-(heteroarylacetamido)-2-oxo-axetidine-1-sulfonic acid and of formula I

Wherein M is a hydrogen or pharmaceutically acceptable salt formingcation; X is CH, N or C-halogen; R is C₁-C₃ alkyl which is unsubstitutedor substituted with at least one of (a) a halogen atom (b) OR₅ whereinR₅ is hydrogen, or, CONH₂, provided that, when R is methyl, it cannot besubstituted with both a halogen atom and OR₅; R₁ and R₂ independentlyare OH, COOH, CONH₂, NH₂, dimethylamino, OCH₃, NO₂, CN, optionallysubstituted phenyl or C₁-C₃ alkyl; or R₁ and R₂ together are—O—CH═CH—CH₂—, —O—CH₂—CH₂—O—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—,—CH═CH—CH═CH— or —CH═C(OH)—C(OH)═CH— which together with the carbonatoms to which they are bound form a 5 membered or six membered cyclicring; and R₃ and R₄ independently are hydrogen, optionally substitutedC₁-C₃ alkyl, optionally substituted phenyl or C₃-C₆ cycloalkyl; or R₃and R₄ together with the C to which they are attached are C₃-C₆cycloalkyl.
 11. The composition, according to claim 8, wherein saidcomposition is formulated for parental use and comprises about 0.1 to 5w/w % of a compound selected from the group consisting of a Syn isomerof a racemate and an optical isomer of 3-(heteroarylacetamido)-2-oxo-axetidine-1-sulfonic acid and of formula I

Wherein M is a hydrogen or pharmaceutically acceptable salt formingcation; X is CH, N or C-halogen; R is C₁-C₃ alkyl which is unsubstitutedor substituted with at least one of (a) a halogen atom (b) OR₅ whereinR₅ is hydrogen, or, CONH₂, provided that, when R is methyl, it cannot besubstituted with both a halogen atom and OR₅; R₁ and R₂ independentlyare OH, COOH, CONH₂, NH₂, dimethylamino, OCH₃, NO₂, CN, optionallysubstituted phenyl or C₁-C₃ alkyl; or R₁ and R₂ together are—O—CH═CH—CH₂—, —O—CH₂—CH₂—O—, —CH₂CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—,—CH═CH—CH═CH— or —CH═C(OH)—C(OH)═CH— which together with the carbonatoms to which they are bound form a 5 membered or six membered cyclicring; and R₃ and R₄ independently are hydrogen, optionally substitutedC₁-C₃ alkyl, optionally substituted phenyl or C₃-C₆ cycloalkyl; or R₃and R₄ together with the C to which they are attached are C₃-C₆cycloalkyl.
 12. The method of claim 9, wherein the therapeuticallyeffective amount of the compound comprises an oral dosage of about 50 to1500 mg/day of the compound for an adult in 2-4 divided doses.
 13. Themethod of claim 9, wherein the therapeutically effective amount of thecompound comprises an injection of about 100 mg of the compound.
 14. Themethod of claim 9, wherein the therapeutically effective amount of thecompound is diluted with physiological saline or glucose injectionliquid.
 15. The method of claim 9, wherein the therapeutically effectiveamount of the compound comprises a suppository containing about 1-1000mg of the compound.
 16. A method of producing a compound of formula I

Wherein M is a hydrogen or pharmaceutically acceptable salt formingcation; X is CH; R is CH₃, CH₂F or CH₂OCONH₂; R₁ is OH; R₂ is Hydrogen;or R₁ and R₂ together are —CH═C(OH)—C(OH)═CH— which, together with thecatons to which R₁ and R₂ are bound, forms a six member cyclic ring; andR₃ and R₄ independently are hydrogen; or R₃ and R₄ together with the Cto which they are attached are cyclopropyl; Wherein said methodcomprises: (a) reacting a 3-amino-azetidine-2-one sulfonic acid offormula (II)

with a heteroaryl carboxylic acid of formula (III)

 and (b) deprotecting the protecting group.
 17. The compound of formulaI as set forth in claim 1, wherein the optical isomer is a pure single Ror S diasteroisomer at an asymmetric carbon atom present in themolecule.
 18. A pharmaceutical composition comprising a compound offormula I

Wherein M is a hydrogen or pharmaceutically acceptable salt formingcation; X is CH; R is CH₃, CH₂F or CH₂OCONH₂; R₁ is OH; R₂ is Hydrogen;or R₁ and R₂ together are —CH═C(OH)—C(OH)═CH— which, together with thecatons to which R₁ and R₂ are bound, forms a six member cyclic ring; andR₃ and R₄ independently are hydrogen; or R₃ and R₄ together with the Cto which they are attached are cyclopropyl.
 19. A method of treating abacterial infection in a subject in need of such treatment comprisingadministering to said subject in need of such treatment atherapeutically effective amount of a compound selected from the groupconsisting of a Syn isomer of a racemate and an optical isomer of3-(heteroaryl acetamido)-2-oxo-axetidine-1-sulfonic acid and of formulaI

Wherein M is a hydrogen or pharmaceutically acceptable salt formingcation; X is CH; R is CH₃, CH₂F or CH₂OCONH₂; R₁ is OH; R₂ is Hydrogen;or R₁ and R₂ together are —CH═C(OH)—C(OH)═CH— which, together with thecatons to which R₁ and R₂ are bound, forms a six member cyclic ring; andR₃ and R₄ independently are hydrogen; or R₃ and R₄ together with the Cto which they are attached are cyclopropyl.